1. Field of the Invention
The present invention relates to a biochemical sensor utilizing an optical thin film, components for the sensor, and a measuring apparatus utilizing the same.
2. Description of the Background
The bindings between biochemical substances, such as in an antigen-antibody reaction, have generally been measured using a “label” such as a radioactive substance or fluorescence materials. Labeling is laborious and, particularly, the labeling of proteins is sometimes complicated in view of the method and the fact that the proteins may be altered by this labeling process. In view of the above, a biochemical sensor utilizing the change of interference color of an optical thin film has been known as a method of directly measuring the binding between biochemical substances in a simple and convenient manner, without using a label.
A biochemical sensor is described in the article of T. Sandstrom, et. al., APPL. OPT., 24, 472, 1985 (hereafter “Non-Patent Document 1”). An example is to be described with reference to the model shown in FIG. 1. An optical thin film 2 is disposed on a substrate 1. The refractive index of air is 1.00, the refractive index of the material for the optical thin film 2 is 1.50, and the refractive index of the substrate 1 is 2.25 in this exemplary sensor. When the thickness of the optical thin film is adjusted or controlled to an optical length corresponding to ¼ (or an odd number multiple thereof) of a wavelength λ0 of visible light (for example, ¾ λ0, 5/4λ0, etc.), the optical thin film acts as an anti-reflection film in which the intensity of reflected light perpendicular to the optical thin film is 0 at a wavelength λ0 as shown by a reflection spectrum A in FIG. 2. Thus, the sensor produces an interference color.
A single molecular layer of a first biochemical substance 3 is disposed on the optical thin film 2. Assuming the biochemical substance as a protein, the refractive index is about 1.5 and the thickness of the layer is about 10 nm. This means that the thickness of the optical thin film increases in terms of optics. Therefore, the reflection spectrum changes from a solid line A to a short dashed line A′ in FIG. 2, and the interference color changes. When a second biochemical substance 4 is biochemically binded to the first biochemical substance 3, the film thickness further increases which causes a change from the short dashed line A′ to the broken line A″ in FIG. 2 and another change in the interference color. Thus, binding of the second biochemical substance 4 may be detected.
As a general detection procedure, the optical thin film 2 on the substrate 1 covered with a single molecular layer 3 of a first biochemical substance is prepared first. This preparation is put into a solution of a second biochemical substance (4). Then, the preparation is taken out of the solution, dried and then the change of the interference color from the short dashed line A′ to broken line A″ in FIG. 2 is examined.
Further, light reflection caused at the back of the substrate 1 may be suppressed by using a light absorbing material, for example silicon, as the material for the substrate 1. Silicon monoxide is vapor deposited as an optical thin film to the silicon substrate and the uppermost surface layer is formed into silicon dioxide of 2 to 3 nm thickness obtained by spontaneous oxidation of silicon monoxide, thereby preparing a chemically stable film.
As described above, in the existing biochemical sensors utilizing an optical thin film disposed on the light absorbing substrate, the interference color is measured after taking the sensor out of solution and drying the sensor in air. Further, Japanese Patent Application JP-A No. 195242/1983 (hereafter “Patent Document 1”) describes detection of a chemical substance using dielectric layers. Patent Document 1 describes that a SiO2 layer is disposed on the surface of a carrier comprised of silicon to form a reflection-reducing coating.
However, since the sensor described in Non-Patent Document 1 above is taken out in the air and the interference color is measured after drying for detection, it takes an undesirable amount of time during the drying step, and improvement for the throughput is desired. Further, since measurement is conducted after a lapse of a predetermined time after the beginning of the reaction, the sensor is sometimes taken out into the air before saturation of the reaction, depending upon the way in which the predetermined time is set, so that measurement cannot always be conducted with high (maximum) accuracy. On the other hand, if a long predetermined time is set in order for measurement to be taken after sufficient saturation of the reaction, because the sensor is dipped into the solution after waiting for the saturation of the reaction, the efficiency is poor in view of time.
At the same time, with respect to the chemical resistance of the sensor, an alkali cleaning is an effective method for removing organic matters that may be deposited on the sensor. Further, when surface modification is applied for immobilization of the first biochemical substance on the sensor surface and for preventing non-specific adsorption of molecules to the sensor surface, a sensor chip is sometimes dipped in an alkali solution, so that alkali resistance is important. However, a silicon substrate has poor resistance to an aqueous alkali solution, and it dissolves in an aqueous 1 M sodium hydroxide while evolving bubbles. Further, using silicon dioxide as the uppermost layer of the optical thin film described in Non-Patent Document 1 has no sufficient resistance to alkali.